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  • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
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  • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
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  • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
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  • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
  • C12Q2563/149—Particles, e.g. beads
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  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
  • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
  • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels

Definitions

• the present invention relates to a method for detecting a target nucleic acid molecule using a photocrosslinking reaction.
• Nucleic acid molecules in a sample solution can be detected by a hybridization method using a labeled probe that specifically hybridizes with the nucleic acid molecule.
• a hybridization method using a labeled probe that specifically hybridizes with the nucleic acid molecule.
• the nucleic acid molecule that has been hybridized and immobilized on the beads is precipitated on the bottom of a container.
• the temperature of the reaction liquid is subsequently gradually raised to a temperature equal to or higher than the denaturation temperature of the target nucleic acid to release the labeling probe from the beads into the supernatant followed by measuring the amount of light or fluorescence in the supernatant over time (see, for example, Japanese Unexamined Patent Application, First Publication No. 2004-121231).
• a method that consists of introducing a reactive functional group into a base that composes an oligonucleotide, and forming covalent bonds between other oligonucleotides and other molecules through this reactive functional group (crosslinking).
• crosslinking examples of technologies for crosslinking nucleic acid molecules by covalent bonding using a base derivative introduced with a reactive functional group include a method that uses 2-amino-6-vinylpurine (see, for example, Sasaki, S., Yakugaku Zasshi, 2002, Vol. 122, No. 12, pp.
• FRET fluorescence resonance energy transfer
• non-specific association products association products formed by non-specific hybridization
• detection of the association product formed is typically carried out under ordinary measurement temperature conditions (such as room temperature)
• the formation of non-specific association products can be effectively suppressed due to stabilization of the association product formed between the FRET probe and target nucleic acid molecule by a photocrosslinking reaction.
• a FRET probe that specifically binds to a specific nucleic acid molecule is also used to detect antigen-antibody reactions.
• a method has been disclosed that consists of carrying out an antigen-antibody reaction using an antibody labeled with single-stranded DNA and binding the antigen-antibody complex formed with a FRET probe complementary to the single-stranded DNA used to label the antibody, followed by releasing the fluorescent substance in the FRET probe from the antigen-antibody complex into the reaction liquid supernatant by treating with a nucleolytic enzyme, and detecting the antigen by measuring fluorescence intensity of the reaction liquid supernatant (see, for example, Japanese Unexamined Patent Application, First Publication No. 2011-033613).
• a method for detecting a target nucleic acid molecule in one aspect of the present invention has:
• the first marker is released from the three-component association product by:
• a reaction for forming the covalent bond is a photochemical reaction mediated by a photoreactive base derivative.
• At least one base in a region of the second nucleic acid probe that hybridizes with the target nucleic acid molecule may be substituted with a photoreactive base derivative.
• the photoreactive base derivative is 3-cyanovinylcarbazole nucleoside
• the covalent bond is formed by irradiating the sample solution with light at 340 nm to 380 nm.
• the three-component association product bound to the solid phase carrier recovered in the (e) is washed with a washing solution having a salt concentration at which the Tm value of the first nucleic acid probe is 25° C. or lower.
• the detecting of the first marker is carried out using a fluorescent single molecule measurement method.
• the detecting of the first marker is carried out by:
• Another aspect of the present invention is a target nucleic acid molecule detection kit used in the method for detecting a target nucleic acid molecule described in any of (1) to (11) above, including:
• a first nucleic acid to which a first marker, that is a luminescent substance, is bound, and which specifically hybridizes with a target nucleic acid molecule
• a second nucleic acid probe to which a second marker is bound, and which specifically hybridizes with a target nucleic acid molecule in a region that differs from the region hybridized by the first nucleic acid probe.
• At least one base in a region of the first nucleic acid probe that hybridizes with the target nucleic acid molecule is substituted with a photoreactive base derivative
• At least one base in a region of the second nucleic acid probe that hybridizes with the target nucleic acid molecule may be substituted with a photoreactive base derivative.
• a solid phase carrier provided with a site that binds with the second marker may be further included.
• FIG. 1 is a drawing schematically showing one aspect of the method for detecting a target nucleic acid molecule of the present invention.
• FIG. 2 is a drawing showing the results of using the FIDA method to measure the number of molecules of the fluorescent substance TAMRA released from magnetic beads recovered from various sample solutions by a nucleolytic enzyme reaction in Example 1.
• FIG. 3 is a drawing showing the results of using the FCS method to measure the number of molecules of the fluorescent substance TAMRA released from magnetic beads recovered from various sample solutions by a nucleolytic enzyme reaction or irradiating with ultraviolet light under low salt concentration conditions in Example 2.
• FIG. 4 is a drawing showing the results of using the FCS method to measure the number of molecules of the fluorescent substance TAMRA released from magnetic beads recovered from various sample solutions by heating under low salt concentration conditions or alkaline conditions in Example 3.
• FIG. 5 is a drawing showing the results of using the FIDA method to measure the number of molecules of the fluorescent substance TAMRA released from magnetic beads recovered from various sample solutions by heating in Example 4.
• the method for detecting a target nucleic acid molecule in one aspect of the present invention has the following operations (a) to (g):
• a target nucleic acid molecule refers to a nucleic acid molecule having a specific base sequence that is a target of detection.
• the target nucleic acid molecule may be a nucleic acid molecule having a base sequence present in the chromosome of an animal or plant or gene of a bacterium or virus, or a nucleic acid molecule having an artificially designed base sequence.
• the target nucleic acid molecule may be a double-stranded nucleic acid or single-stranded nucleic acid.
• it may be DNA or RNA.
• examples of the aforementioned target nucleic acid molecule include microRNA, siRNA, mRNA, hnRNA, genomic DNA, synthetic DNA obtained by PCR or other amplification and cDNA synthesized from RNA using a reverse transcriptase.
• the nucleic acid-containing sample is a sample that contains a nucleic acid molecule.
• the aforementioned nucleic acid-containing sample include biological samples collected from animals and the like, samples prepared from cultured cells and the like, and reaction solutions following a nucleic acid synthesis reaction.
• a biological sample may be used as is for the nucleic acid-containing sample, or the nucleic acid-containing sample may be a nucleic acid solution that has been extracted or purified from a biological sample and the like.
• nucleic acid probe that specifically hybridizes with a target nucleic acid molecule refers to that which is only required to be a nucleic acid probe that preferentially hybridizes with a target nucleic acid molecule rather than binding to another nucleic acid molecule having a similar base sequence with that the target nucleic acid molecule, and is not required to not hybridize at all with nucleic acid molecules other than the target nucleic acid molecule.
• the nucleic acid probe that specifically hybridizes with a target nucleic acid molecule may be an oligonucleotide having a base sequence completely complementary to a partial base sequence of the target nucleic acid molecule, or may have a base sequence that contains one to several base mismatches with a partial base sequence of the target nucleic acid molecule.
• the first nucleic acid probe used in the present embodiment has a first marker bound thereto and specifically hybridizes with a target nucleic acid molecule.
• the first nucleic acid probe include an oligonucleotide that has a base sequence completely complementary to a partial base sequence of a target nucleic acid molecule or a base sequence having one to several base mismatches with that partial base sequence, and has the first marker bound thereto.
• the first marker is a luminescent substance.
• luminescent substances include particles that emit light by fluorescence, phosphorescence, chemiluminescence, bioluminescence or light scattering (and are normally molecules or aggregates thereof).
• a fluorescent substance may be used for the first marker due to high detection sensitivity.
• the fluorescent substance is a substance that releases fluorescent light as a result of being irradiated with light of a specific wavelength, and can be suitably selected and used from among fluorescent dyes, quantum dots and the like used in optical analyses such as FCS or FIDA.
• the second nucleic acid probe used in the present embodiment has a second marker bound thereto and specifically hybridizes with a target nucleic acid molecule.
• the second nucleic acid probe include an oligonucleotide that has a base sequence completely complementary to a partial base sequence of a target nucleic acid molecule or a base sequence having one to several base mismatches with that partial base sequence, and has the second marker bound thereto.
• the second marker is a substance that can be detected by being distinguished from the first marker.
• the second marker may be a non-luminescent substance or a luminescent substance that has luminescence properties differing from those of the first marker.
• different luminescent properties refer to differences in light intensity at a specific wavelength (such as having different fluorescence intensity at a specific wavelength).
• Examples of the second marker include fluorescent substances having different luminescence properties from those of the first marker, nucleic acids (oligonucleotides), hydrophilic organic compounds, biotin, glutathione, dinitrophenol (DNP), digoxigenin (Dig), digoxin, sugar chains composed of two or more sugars, polypeptides composed of six or more amino acids, auxins, gibberellins, steroids, proteins and analogs thereof.
• An oligonucleotide composing the first nucleic acid probe or second nucleic acid probe may be DNA or RNA, may be artificially amplified in the manner of cDNA, or may contain all or a portion of a nucleic acid-like substance capable of forming a nucleotide chain and base pairs in the same manner as naturally-occurring nucleic acid bases.
• nucleic acid-like substances include substances in which side chains and the like of naturally-occurring nucleotides (nucleotides present in nature) in the manner of DNA or RNA have been modified by functional groups such as an amino group, and substances that have been labeled with a protein or low molecular weight compound and the like.
• nucleic acid-like substances include bridged nucleic acids (BNA), nucleotides in which an oxygen atom at position 4′ of a naturally-occurring nucleotide has been substituted with a sulfur atom, nucleotides in which a hydroxyl group at position 2′ of a naturally-occurring nucleotide has been substituted with a methoxy group, hexitol nucleic acids (HNA) and peptide nucleic acids (PNA).
• BNA bridged nucleic acids
• HNA hexitol nucleic acids
• PNA peptide nucleic acids
• An oligonucleotide that composes the first nucleic acid probe or the second nucleic acid probe may have a region other than the region that hybridizes with the target nucleic acid molecule.
• a region that hybridizes with a target nucleic acid molecule and a region that binds the first marker or the second marker may be linked with a linker having a suitable base length.
• the first nucleic acid probe or the second nucleic acid probe can be produced by designing a base sequence based on base sequence information of a target nucleic acid molecule or base sequence information of a region that forms a base pair, and binding a marker to the nucleic acid probe synthesized based on that design.
• a marker may also be bound simultaneous to synthesis of the nucleic acid probe.
• Design and synthesis of the first nucleic acid probe or the second nucleic acid probe, a binding reaction between the first nucleic acid probe and the first marker, and a binding reaction between the second nucleic acid probe and a second marker can be carried out according to ordinary methods.
• Both the first nucleic acid probe and the second nucleic acid probe hybridize with a single target nucleic acid molecule, and a three-component association product is formed that is composed thereof. Namely, a region that hybridizes with the first nucleic acid probe and a region that hybridizes with the second nucleic acid probe in a target nucleic acid molecule are mutually different.
• the target nucleic acid molecule is a double-stranded nucleic acid molecule
• both the first nucleic acid probe and the second nucleic acid probe hybridize with a single-stranded nucleic acid molecule thereof.
• the first nucleic acid probe and the second nucleic acid probe may hybridize simultaneously with a target nucleic acid molecule.
• Conditions for specific association between the first nucleic acid probe and a target nucleic acid molecule and conditions for specific association between the second nucleic acid probe and a target nucleic acid molecule may be nearly the same conditions in order to allow the first nucleic acid probe and the second nucleic acid probe to hybridize with a single target nucleic acid molecule.
• the specific association conditions are dependent on such factors as the types and lengths of the base sequences of the target nucleic acid molecule and nucleic acid probes.
• the first nucleic acid probe and the second nucleic acid probe may be designed so as to satisfy specific association conditions.
• specific association conditions between a target nucleic acid molecule and the nucleic acid probes can be determined from a melting curve. Since the formation of an association product is typically dependent on temperature conditions and salt concentration conditions, a melting curve can be determined by changing the temperature of a solution containing only a nucleic acid probe and a target nucleic acid molecule from a high temperature to a low temperature and measuring optical absorbance of the aforementioned solution. Temperature conditions over a range from the temperature at which a nucleic acid probe present in the form of a single strand begins to form an association product with a target nucleic acid molecule to the temperature at which nearly all have formed an association product as determined from the resulting melting curve can be taken to be the specific association conditions. Specific association conditions can also be determined by similarly determining a melting curve by changing the salt concentration in the solution from a low concentration to a high concentration instead of changing temperature.
• Tm value melting temperature
• the Tm value of a region having a base sequence complementary to a target nucleic acid molecule can be calculated from base sequence information of a nucleic acid probe by using commonly used primer/probe design software and the like.
• Conditions in which the temperature is in the vicinity of the Tm value can be used as specific association conditions. More detailed specific association conditions can be determined by experimentally determining a melting curve at a temperature in the vicinity of the calculated Tm value.
• a sample solution is prepared by adding a nucleic acid-containing sample, a first nucleic acid probe and a second nucleic acid probe to a suitable solvent.
• a suitable solvent there are no particular limitations on the aforementioned solvent provided it is a solvent that does not damage the first marker or second marker, and can be suitably selected and used from among buffers commonly used in the art. Examples of such buffers include phosphate buffers and Tris buffers, such as phosphate-buffered saline (PBS, pH 7.4).
• an organic solvent such as formamide may also be used depending on the type of first marker and second marker.
• nucleic acid molecules in the prepared sample solution are denatured.
• denaturing of nucleic acid molecules refers to the dissociation of base pairs. For example, this refers to converting a double-stranded nucleic acid to a single-stranded nucleic acid.
• luminescent substances such as fluorescent substances
• denaturation by high-temperature treatment (heat denaturation) or denaturation by low salt concentration treatment can be carried out.
• Heat denaturation is a simple procedure. Nucleic acid molecules in a sample solution can be denatured by subjecting the sample solution to high-temperature treatment.
• nucleic acid molecules can be denatured by incubating at a temperature of 90° C. for DNA or 70° C. for RNA for several seconds to about 2 minutes, since the denaturation temperature varies according to the base length of the target nucleic acid molecule, the aforementioned incubation temperature is not limited thereto provided denaturation is possible.
• denaturation by low salt concentration treatment can be carried out by, for example, adjusting the salt concentration of the sample solution to be sufficiently low by diluting with purified water and the like.
• nucleic acid molecules in the aforementioned sample solution are allowed to associate.
• Formation of an association product of a target nucleic acid molecule, first nucleic acid probe and second nucleic acid probe is carried out under specific association conditions. More specifically, in the case of having carried out heat denaturation, nucleic acid molecules in the aforementioned sample solution are allowed to suitably associate by lowering the temperature of the sample solution to a temperature that satisfies specific association conditions after high-temperature treatment. The temperature of the sample solution can be lowered to a temperature that is within about ⁇ 3° C. of the Tm value of a region in the first nucleic acid probe and second nucleic acid probe having a base sequence complementary to the target nucleic acid molecule.
• nucleic acid molecules in the sample solution are allowed to suitably associate by similarly raising the salt concentration of the sample solution to a concentration that satisfies specific association conditions by adding a salt solution and the like after low salt concentration treatment.
• the temperature of the solution can be lowered comparatively slowly when forming an association product.
• the liquid temperature of the solution can be lowered at a temperature lowering rate of 0.05° C./second or higher.
• a surfactant in order to suppress non-specific hybridization, a surfactant, formamide, dimethylsulfoxide or urea and the like may be added to the aforementioned solution in advance.
• At least one covalent bond is formed between the target nucleic acid molecule and the first nucleic acid probe and at least one covalent bond is formed between the target nucleic acid molecule and the second nucleic acid probe in the three-component association product formed in the (c) composed of the target nucleic acid molecule, the first nucleic acid probe and the second nucleic acid probe.
• the three-component association product consisting of the target nucleic acid molecule, the first nucleic acid probe and the second nucleic acid probe can be stabilized even in a state in which temperature is not controlled to so-called normal temperature.
• formation of covalent bonds in (d) may be carried out in a state in which the three-component association product formed in (c) is maintained. For example, in the case of having formed the three-component association product in (c) by lowering the temperature of the sample solution to a temperature that allows formation of the three-component association product, formation of covalent bonds in (d) may be carried out without changing the temperature of the sample solution.
• the same conditions as those during formation of the three-component association product in (c) may be the conditions under which the temperature and salt concentration of the sample solution are mutually the same, the conditions are not necessarily required to be completely physically identical provided the ease of forming the association product of the target nucleic acid molecule, the first nucleic acid probe and the second nucleic acid probe, and the ease of forming an association product consisting of a nucleic acid molecule other than the target nucleic acid molecule, the first nucleic acid probe and the second nucleic acid probe, are substantially the same when forming the three-component association product in (c) and when forming covalent bonds in (d).
• covalent bonds may be formed by a photochemical reaction.
• a photochemical reaction refers to a reaction that is carried out by radiating light of a specific wavelength and then utilizing that light energy. Since a method that forms covalent bonds by a photochemical reaction is able to form covalent bonds between nucleic acid strands of an association product by irradiating a sample solution with light of a specific wavelength, it is not necessary to vary conditions such as the composition of the aforementioned sample solution. Consequently, effects on the association product in the sample solution other than the formation of covalent bonds can be suppressed and the procedure is simple.
• first nucleic probe in which at least one base in a region of the first nucleic acid probe that hybridizes with a target nucleic acid molecule is substituted with a photoreactive base derivative
• second nucleic acid probe in which at least one base in a region of the second nucleic acid probe that hybridizes with a target nucleic acid molecule is substituted with a photoreactive base derivative
• covalent bonds mediated by the aforementioned photoreactive base derivative can be formed between the target nucleic acid molecule and the first nucleic acid probe and between the target nucleic acid and the second nucleic acid probe by a photochemical reaction.
• bases substituted with photoreactive base derivatives in the first nucleic acid probe and the second nucleic acid probe are bases in regions that hybridize with the target nucleic acid molecule.
• only one base may be substituted with a photoreactive base derivative or two or more bases may be substituted with photoreactive base derivatives.
• a photoreactive base derivative refers to a base derivative that has a site at which the reactivity of an organic synthesis reaction (photoreactive site) is activated as a result of being irradiated with light of a specific wavelength, and is able to form a nucleic acid chain in the same manner as a naturally-occurring nucleotide.
• a photoreactive base derivative is 3-cyanovinylcarbazole nucleoside ( CNV K) (see, for example, Non-Patent Document 2 or Non-Patent Document 3).
• CNV K 3-cyanovinylcarbazole nucleoside
• a nucleic acid probe in which a base has been substituted with a photoreactive base derivative can be produced by using an unsubstituted nucleic acid probe as a raw material when, for example, synthesizing a nucleic acid probe using a known oligonucleotide synthesizer.
• a substituted nucleic acid probe can be obtained by introducing a suitable photoreactive functional group into a base that composes the nucleic acid probe by a known organic synthesis reaction.
• CNV K for the photoreactive base derivative
• a first nucleic acid probe and a second nucleic acid probe in a sample solution in which they are contained, when the sample solution is irradiated with ultraviolet light containing light of 300 nm to 380 nm, light of 340 nm to 380 nm, light of 360 nm to 370 nm, or light of 366 nm, covalent bonds are formed between atoms that compose the pyrimidine base in the target nucleic acid molecule that forms a base pair with a purine base adjacent to the 5′-side of CNV K and atoms that compose CNV K.
• a photoreactive base derivative obtained by adding psoralen to thymine (T) or adenine (A) through a linker may also be used.
• T thymine
• A adenine
• a linker for example, Proc. Natl. Acad. Sci. USA, Vol. 88, pp. 5602-5606, July 1991.
• a psoralen-bound nucleic acid probe is prepared in which psoralen is bound to the T or A in the aforementioned TA sequence through a linker.
• the first nucleic acid probe or the second nucleic acid probe that forms a base pair through this psoralen is crosslinked with the target nucleic acid molecule, thereby stabilizing the three-component association product.
• a solid phase carrier provided with a site that binds with a second marker is first added to the aforementioned sample solution, and the solid phase carrier and the three-component association product are bound through the second marker in the three-component association product. Subsequently, the three-component association product bound to the solid phase carrier is recovered by solid-liquid separation treatment.
• the solid phase carrier used in the present embodiment is provided with a site that binds with a second marker.
• the solid phase carrier is a solid phase carrier having on the surface thereof a substance that specifically or non-specifically binds or absorbs to the second marker.
• the second marker is an oligonucleotide
• examples of the aforementioned substance include an oligonucleotide that hybridizes with the aforementioned oligonucleotide, an antigen or antibody to the second marker, a ligand or receptor for the second marker, and a substance that specifically binds with the second marker in the manner of biotin and avidin.
• the solid phase carrier may non-specifically bind to the second marker, it may bind specifically from the viewpoint of greater accuracy in detecting or quantifying target nucleic acid molecules.
• the substance that specifically or non-specifically binds or adsorbs to the second marker may be physically adsorbed or chemically bound to a functional group on the surface of the solid phase carrier.
• the aforementioned substance can be bound by a method suitable for each functional group.
• Examples thereof include a reaction in which 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC), a reaction in which 1-ethyl-3-(3-dimethylaminopropyl)carbodiimido hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are mixed in advance followed by bonding between carboxylic acid and an amino group, a reaction in which amino groups are crosslinked using a bipolar linker, and a reaction in which an activated aldehyde group or tosyl group bonds with a functional group in the substance that specifically or non-specifically binds or adheres to the second marker.
• a functional group may be preliminarily coated on the surface of the solid phase carrier in the case the surface of the solid phase carrier does not have a functional group.
• the solid phase carrier is a solid provided with a site that binds with the second marker.
• it may be a particle such as a bead that can be suspended in water and can be separated from a liquid by a common solid-liquid separation procedure, or a membrane, container or chip substrate.
• Specific examples of solid phase carriers include magnetic beads, silica beads, agarose gel beads, polyacrylamide resin beads, latex beads, plastic beads, ceramic beads, zirconia beads, silica membranes, silica filters and plastic plates.
• the solid phase carrier consists of particles such as beads, the aforementioned solid phase carrier is added to the aforementioned sample solution.
• the sample solution is passed through the solid phase carrier.
• the solid phase carrier is a container coated with a substance that binds with the second marker on the inner walls thereof, the sample solution is injected into the container serving as the solid phase carrier.
• the second marker is biotin
• beads or filter having avidin or streptavidin bound to the surface thereof can be used as a solid phase carrier.
• the second marker is digoxigenin (Dig)
• beads or filter having anti-Dig antibody bound to the surface thereof can be used as a solid phase carrier.
• the aforementioned three-component association product is allowed to bind to the solid phase carrier through the second marker in the three-component association product by contacting the solid phase carrier with a sample solution containing the three-component association product.
• the three-component association product bound to the solid phase carrier can be separated from the free first nucleic acid probe present in the liquid phase and recovered.
• the solid-liquid separation procedure is a method that allows separation and recovery of the solid phase carrier in solution from a liquid component, and can be suitably selected and used from among known treatments used for solid-liquid separation treatment.
• a suspension containing the solid phase carrier may be subjected to centrifugal separation treatment to precipitate the solid phase carrier followed by removal of the supernatant, or the aforementioned solution may be filtered using a filter followed by recovering the solid phase carrier remaining on the surface of the filter paper.
• a magnet may be brought in close proximity to a container containing the aforementioned solution to cause the solid phase carrier to converge at the surface of the container closest to the magnet followed by removing the supernatant.
• the solid phase carrier is a container having an inner wall coated with a substance that binds to the second marker, liquid inside the container serving as the solid phase carrier is discarded.
• the solid phase carrier is a membrane or filter, binding between the solid phase carrier and three-component association product and separation and recovery of the three-component association product bound to the solid phase carrier from the free first nucleic acid probe can be carried out in a single procedure by passing the aforementioned sample solution through the aforementioned solid phase carrier.
• a solid phase carrier provided with a site that binds with a second marker is preliminarily added to a sample solution together with a nucleic acid-containing sample, a first nucleic acid probe and a second nucleic acid probe in (a), and in (c), after having formed a three-component association product bound to the solid phase carrier, the three-component association product bound to the solid phase carrier may be separated and recovered from the free first nucleic acid probe by solid-liquid separation treatment.
• the three-component association product bound to the solid phase carrier may be separated and recovered from the free first nucleic acid probe by solid-liquid separation treatment.
• the second marker used at this time may be reversibly or irreversibly bound to the solid phase carrier.
• a solution containing the three-component association product bound to the solid phase carrier is prepared by adding a suitable solvent to the recovered solid phase carrier.
• the recovered solid phase carrier is supplied to (f) in the form of a solution in which it is contained therein.
• a suitable solvent there are no particular limitations on the aforementioned solvent provided it is a solvent that does not hinder detection of light released from the first marker in a subsequent, and can be suitably selected and used from among buffers commonly used in the art.
• the aforementioned buffers include phosphate buffers and Tris buffers, such as phosphate-buffered saline (PBS, pH 7.4).
• the recovered solid phase carrier may be washed with a suitable solvent prior to (f). As a result of washing, the free first nucleic acid probe can be more completely separated and removed from the three-component association product bound to the solid phase carrier.
• a suitable solvent there are no particular limitations on the solvent used to wash the solid phase carrier provided it does not impair binding between the second marker and the solid phase carrier, and may the same as or different from the buffer used to prepare the solution containing the three-component association product bound to the solid phase carrier supplied to (f).
• the solid phase carrier may be washed in a state in which the formation of non-specific association products (such as association products with the second nucleic acid probe) by the first nucleic acid probe is suppressed.
• the solid phase carrier is washed with a solvent having a salt concentration such that the Tm value of the first nucleic acid probe is extremely low.
• the solid phase carrier may be washed with a solvent having a salt concentration such that the Tm value of the first nucleic acid probe is lower than the temperature during washing.
• a solvent may be used for washing that has a salt concentration such that the Tm value of the first nucleic acid probe is 25° C. or lower.
• Specific examples of the aforementioned solvent include solutions having a low salt concentration used during washing in a hybridization method, such as solutions having salt concentration of ⁇ 0.01 SSC (1.5 mM NaCl or 0.15 mM sodium citrate solution) or lower.
• the aforementioned solvent may also be a solvent that does not contain salt such as water.
• washing with a highly stringent solution makes it possible to suppress the formation of non-specific association products and effectively remove the free first nucleic acid probe.
• the three-component association product can be stably maintained even if washed under highly stringent conditions.
• the first marker can be released by adding a nucleolytic enzyme to the solution containing the three-component association product bound to the solid phase carrier and decomposing the first nucleic acid probe and other nucleic acid molecules with the aforementioned enzyme (f2).
• a nucleolytic enzyme used to release the first marker, and may be a DNA nucleolytic enzyme, RNA nucleolytic enzyme or restrictase.
• DNA nucleolytic enzymes include S1 nuclease, mung bean nuclease, BAL 31 nuclease, exonuclease I, exonuclease III and DNase I etc.
• RNA nucleolytic enzymes include ribonuclease H etc.
• examples of DNA/RNA nucleolytic enzymes include micrococcal nuclease etc.
• the first nucleic acid probe or the second nucleic acid probe may be designed so as to contain a restrictase recognition sequence corresponding to a restrictase within the region in the first nucleic acid probe or second nucleic acid probe that hybridizes with the target nucleic acid molecule.
• the first marker can be separated from the solid phase carrier by digesting the restrictase recognition site in the three-component association product by restrictase treatment.
• restrictases include, but are not limited to, EcoRI and HindIII etc.
• the first marker can also be released by chemically decomposing the first nucleic acid probe and other nucleic acid molecules with an alkali instead of by an enzymatic reaction (f4). More specifically, after adding a base to a solution containing the three-component association product bound to the solid phase carrier to make the pH of the solution alkaline, the solution is heated to 50° C. to 100° C., 70° C. to 100° C. or 80° C. to 100° C. Alkaline treatment is carried out at a pH and concentration at which the nucleic acid molecules decompose.
• there are no particular limitations on the type of base used and may be an inorganic base or organic base.
• a strongly basic solution such as a 0.1 mM sodium hydroxide solution or 0.1 mM potassium hydroxide solution is added to raise the pH of the solution containing the three-component association product bound to the solid phase carrier to pH 10 or higher, or pH 12 or higher, followed by heating to 50° C. or higher, or 70° C. or higher.
• covalent bonds formed by a photochemical reaction using CNV K have been recently determined to be easily broken by intense energy.
• the energy applied at this time may be light energy, or heat energy.
• the first marker can be released by irradiating the three-component association product bound to the solid phase carrier with ultraviolet light at 300 nm to 380 nm, or 340 nm to 380 nm under dissociating conditions (f1).
• dissociating conditions refer to conditions under which the target nucleic acid molecule and the first nucleic acid probe or the target nucleic acid molecule and the second nucleic acid probe dissociate in the case of not forming covalent bonds between the target nucleic acid molecule and the first nucleic acid probe or between the target nucleic acid molecule and the second nucleic acid probe.
• An example of dissociating conditions consists of low salt concentration conditions such that the Tm value of the first nucleic acid probe is extremely low in the same manner as washing treatment. More specifically, after having added the three-component association product bound to the solid phase carrier to, for example, a solution having a salt concentration such that the Tm value of the first nucleic acid probe is 25° C. or lower (which may also be a solution not containing salt such as water), the solution is irradiated with ultraviolet light at 300 nm to 380 nm.
• the first marker can also be released by heating the three-component association product bound to the solid phase carrier to a temperature sufficiently higher than the Tm value of the first nucleic acid probe. More specifically, the three-component association product bound to the solid phase carrier is heated to 80° C. or higher (f3).
• the three-component association product bound to the solid phase carrier may be heated after having added thereto a solvent such as water that does not impair detection of light released from the first marker.
• the target nucleic acid molecule is detected by detecting the free first marker.
• a single molecule of the first marker is released from a single molecule of the three-component association product. Consequently, the number of first markers detected in (g) is theoretically equal to the number of target nucleic acid molecules in the nucleic acid-containing sample added in (a).
• the free first marker can be detected by irradiating with light of an optimum wavelength in terms of the optical properties thereof and detecting the optical properties of light emitted from the aforementioned marker. Furthermore, detecting the optical properties of the marker refers to detecting an optical signal of a specific wavelength emitted from the aforementioned marker. Examples of the aforementioned signal include fluorescence intensity and fluorescence polarization.
• the method used to detect the first marker is a method that enables detection and analysis of the intensity of fluorescent signals of molecules present in the solution or chronological changes (fluctuations) thereof.
• the fluorescence intensity emitted from all fluorescent molecules in the solution may be measured or fluorescence intensity may be measured for each molecule.
• the fluorescence intensity of a solution can be measured in accordance with ordinary methods using a fluorescence plate reader or other type of fluorescence spectrophotometer.
• the fluorescence intensity of a solution is dependent upon the amount of the first marker contained in the aforementioned solution. Consequently, by preliminarily preparing a calibration curve indicating the relationship between the content of the first marker and fluorescence intensity, the amount of the first marker in the solution, namely the amount of target nucleic acid molecules in the nucleic acid-containing sample, can be quantified.
• fluorescence correlation spectroscopy FCS
• FIDA fluorescence intensity distribution analysis
• detection and analysis of chronological changes in fluorescence signals of such molecules can be carried out in accordance with ordinary methods using, for example, a known single molecule fluorescence analysis system such as the MF20 Single Molecule Fluorescence Spectroscopy System (Olympus Corp.).
• a target nucleic acid molecule can be detected and analyzed by FIDA by detecting fluctuations in fluorescence intensity of a molecule present in the focused region of a confocal optical system, followed by calculating the number of molecules of the free first marker by carrying out statistical analyses.
• a target nucleic acid molecule can be detected and analyzed by FCS by detecting fluctuations in fluorescence intensity of a molecule present in the focused region of a confocal optical system, followed by calculating the number of molecules of the free first marker by carrying out statistical analyses.
• a fluorescent molecule in a solution can also be detected according to the scanning molecule counting method. More specifically, by moving the location of a photodetection region of an optical system of a confocal microscope or multi-photon microscope in a measurement sample solution using that optical system while detecting fluorescent light from the aforementioned photodetection region, the number of molecules of the free first marker present in the measurement sample solution can be calculated (see, for example, International Publication No. WO 11/108,369, International Publication No. WO 11/108,370 and International Publication No. WO 11/108,371).
• the scanning molecule counting method is a technology that enables counting of particles that emit light (luminescent particles) in a measurement sample solution, or acquisition of information relating to concentration or number density of luminescent particles in a measurement sample solution, by detecting light emitted from the luminescent particles in a microregion when the luminescent particles are dispersed and move about randomly in the measurement sample solution while scanning the interior of the sample solution with the microregion.
• the amount of sample required for measurement is extremely small (such as roughly several tens of microliters) and measurement time is short in the same manner as optical analysis technologies such as FIDA, while also enabling quantitative detection of the concentration, number density or other properties of luminescent particles at a lower concentration or number density in comparison with the case of using an optical analysis technology such as FIDA.
• the “photodetection region” of a confocal microscope or multi-photon microscope refers to a microregion in which light is detected in those microscopes, and in the case illumination light is imparted from an object lens, the region where that illumination light is focused corresponds to a microregion. Furthermore, this microregion is defined by the positional relationship between the object lens and pinhole in a confocal microscope in particular.
• the photodetection mechanism per se of the scanning molecule counting method is composed so as to detect light from a photodetection region of a confocal microscope or multi-photon microscope in the same manner as in the case of optical analysis technologies such as FIDA, the amount of sample solution is similarly extremely small.
• the optical analysis technology of the scanning molecule counting method can be applied to samples solutions in which particle number density or concentration is much lower than the level required by optical analysis technologies such as FIDA.
• the scanning molecule counting method detects individual particles dispersed or dissolved in a solution
• counting of particles, determination of particle concentration or number density in a measurement sample solution, or acquisition of information relating to concentration or number density can be carried out quantitatively using that information.
• particles are detected one at a time by creating a 1:1 correlation between a particle passing through a photodetection region and a detected optical signal, particles dispersed and moving randomly in a solution can be counted. Consequently, the concentration or number density of particles in a measurement sample solution can be determined more accurately than in the past.
• the first marker can be detected even if the concentration of the first marker in the measurement sample solution is even lower than the concentration able to be determined based on fluorescence intensity as measured with a fluorescence spectrophotometer or plate reader.
• Use of the scanning molecule counting method enables target nucleic acid molecules to be quantitatively detected without having to amplify in advance even in the case the concentration of the target nucleic acid molecules in the nucleic acid-containing sample is extremely low and the concentration of the free first marker is 1 femtomole or less.
• the interior of a measurement sample solution is scanned with a photodetection region by changing the light path of the optical system
• the interior of the measurement sample solution can be observed uniformly or the measurement sample solution can be observed in a mechanically stable state without imparting mechanical vibrations or actions attributable to fluid dynamics to the measurement sample solution. Consequently, for example, the reliability of quantitative detection results is improved in comparison with the case of causing the generation of flow in a sample, and measurements can be carried out in the absence of effects caused by dynamic action or artifacts on particles to be detected in the measurement sample solution (the free first marker in the present invention).
• FIG. 1 is a drawing schematically showing one aspect of the method for detecting a target nucleic acid molecule of the present embodiment.
• a fluorescent substance is used for the first marker
• biotin is used for the second marker
• avidin beads are used for the solid phase carrier.
• Covalent bonds are formed by a photochemical reaction using CNV K between the first nucleic acid probe and the target nucleic acid molecule and between the second nucleic acid probe and the target nucleic acid molecule, and the first marker is released using a nucleolytic enzyme.
• a three-component association product formed by hybridization of a target nucleic acid molecule 1 , a first nucleic acid probe 2 having a fluorescent substance 2 a bound thereto, and a second nucleic acid probe 3 having biotin 3 a bound thereto is irradiated with ultraviolet light at 365 nm to respectively form covalent bonds (crosslinks) between CNV K 2 b in the first nucleic acid probe 2 and the target nucleic acid molecule 1 and between CNV K 3 b in the second nucleic acid probe 3 and the target nucleic acid molecule 1 (first illustration in FIG. 1 ).
• Avidin beads 4 are added to the sample solution after crosslinking and the three-component association product is bound to the avidin beads 4 through the biotin 3 a (second illustration in FIG. 1 ). Subsequently, after washing with a low salt concentration solution, the target nucleic acid molecule 1 , the first nucleic acid probe 2 and the second nucleic acid probe 3 are decomposed by treating with a nucleolytic enzyme (third illustration in FIG. 1 ), and the fluorescent substance 2 a is released from the avidin beads 4 (fourth illustration in FIG. 1 ).
• the second nucleic acid probe in the case of enabling the second nucleic acid probe to hybridize with the target nucleic acid molecule in the solid-liquid separation treatment of (e) and the washing treatment prior thereto as well by making the Tm value of the association product of the second nucleic acid probe and the target nucleic acid molecule to be sufficiently higher than the Tm value of the association product of the first nucleic acid probe and the target nucleic acid molecule, at least one covalent bond is formed only between the first nucleic acid probe and the target nucleic acid molecule.
• the target nucleic acid molecule can be detected without being affected by free labeled probe or solid phase carrier in the same manner as the method for detecting a target nucleic acid molecule of the present embodiment.
• the free first nucleic acid probe can be removed by the aforementioned washing treatment.
• An example of a method used to make the Tm value of the association product of the second nucleic acid probe and the target nucleic acid molecule higher than the Tm value of the association product of the first nucleic acid probe and the target nucleic acid molecule consists of composing the second nucleic acid probe so as to contain in at least a portion thereof a nucleic acid-like substance capable of forming a stronger base pair than a naturally-occurring oligonucleotide such as PNA, and composing the nucleic acid chain portion of the first nucleic acid probe with only a naturally-occurring oligonucleotide.
• release of the aforementioned first marker from the three-component association product recovered in the aforementioned (f) can be carried out by washing the three-component association product bound to the solid phase carrier under highly stringent solution conditions to the extent that the second nucleic acid probe is unable to hybridize with the target nucleic acid molecule.
• the first marker forming an association product with the target nucleic acid molecule is released from the solid phase carrier as a result of the second nucleic acid probe bound to the solid phase carrier dissociating from the target nucleic acid molecule.
• kits Various reagents and instruments, including the first nucleic acid probe and the second nucleic acid probe, used in the method for detecting a target nucleic acid molecule of the present embodiment may also be incorporated in a kit.
• the aforementioned kit allows the method for detecting a target nucleic acid molecule of the present invention to be carried out easily.
• a solid phase carrier provided with a site that binds to a second marker, various buffers used to prepare sample solutions, a washing solution for washing the three-component association product after stabilizing with covalent bonds, and an incubator equipped with a constant temperature device, can be included in the aforementioned kit.
• Unlabeled target nucleic acid molecules were detected according to the method for detecting a target nucleic acid molecule of one aspect of the present invention by using, as the target nucleic acid molecule, a single-stranded RNA having a sequence homologous with human microRNA in the form of let-7a (hsa-let-7a, 5′-UGAGGUAGUAGGUUGUAUAGUU-3′)(SEQ ID NO: 1).
• a nucleic acid probe (B-7aL1,5′-TTTTTTTTTTAACTAKACAACCT-3′)(SEQ ID NO: 5) having a base sequence complementary to the region of let-7a from the 3′-terminal to the 13th base on the 3-terminal side, and obtained by substituting a base complementary to the 6th base from the 3′-terminal of let-7a with a crosslinking base derivative in the form of CNV K, and adding a second marker in the form of ten thymidine bases and biotin to the 5′-terminal side, was used as the second nucleic acid probe.
• “K” refers to CNV K.
• the base sequences of each probe prior to substitution with CNV K are shown in SEQ ID NO: 2 and SEQ ID NO: 3.
• the first nucleic acid probe and the second nucleic acid probe were synthesized by Fasmac Co., Ltd.
• Solutions obtained by respectively adding the first nucleic acid probe and the second nucleic acid probe at a final concentration of 100 nM, and respectively adding a target nucleic acid molecule in the form of synthetic RNA composed of abase sequence complementary to let-7a at final concentrations of 10 nM, 1 nM, 100 pM, 10 pM, 1 pM and 0 pM were prepared in 50 ⁇ l aliquots for use as sample solutions (150 mM NaCl, 10 mM Tris-HCl, 0.1% Tween 20).
• An RNAase inhibitor (trade name: SUPERase In, Ambion Corp.) was added to each solution at a concentration of 0.1 U/ ⁇ L.
• the aforementioned target nucleic acid molecule used was synthesized by Hokkaido System Science Co., Ltd.
• Each sample solution was denatured for 2 minutes at 70° C. followed by carrying out hybridization (association) by lowering the temperature to 10° C. at the rate of 1° C./15 seconds. Subsequently, the sample solutions were irradiated with ultraviolet light at 365 nm while cooling in ice. 10 ⁇ L of magnetic beads in the form of Dynabeads (trade name: Dynabeads MyOne Streptavidin, Invitron Ltd.) were added to each sample solution followed by incubating for 15 minutes at room temperature. Subsequently, the magnetic beads were washed once each with ⁇ 1 SSC, ⁇ 0.1 SSC and ⁇ 0.01 SSC.
• Dynabeads trade name: Dynabeads MyOne Streptavidin, Invitron Ltd.
• FIG. 2 The results of using FIDA to measure the number of molecules of the fluorescent substance TAMRA released from the magnetic beads recovered from each sample solution by a nucleolytic enzyme reaction are shown in FIG. 2 .
• Target nucleic acid molecule concentration (nM) is plotted on the horizontal axis, while the number of fluorescent molecules is plotted on the vertical axis.
• the number of fluorescent molecules was determined to decrease concentration-dependently from 10 nM to 1 pM. Fluorescent molecules were determined to be able to be detected at a concentration of 1 pM (50 attomol at 50 ⁇ L) in particular.
• the release of a first marker from a solid phase carrier was carried out in the method for detecting a target nucleic acid molecule in another aspect of the present invention by a nucleolytic enzyme reaction or irradiating with ultraviolet light under low salt concentration conditions.
• samples solutions containing a target nucleic acid molecule, first nucleic acid probe and second nucleic acid probe were prepared in the same manner as Example 1 with the exception of using, as the target nucleic acid molecule, a synthetic RNA composed of a base sequence homologous to let-7a at a final concentration of 10 nM or 0 nM, and after denaturing followed by hybridization (association), covalent bonds were formed by irradiating with ultraviolet light.
• 10 ⁇ L of magnetic beads in the form of Dynabeads (trade name: Dynabeads MyOne Streptavidin, Invitron Ltd.) were added to each sample solution followed by incubating for 15 minutes at room temperature. Subsequently, the magnetic beads were washed once each with ⁇ 1 SSC, ⁇ 0.1 SSC and ⁇ 0.01 SSC.
• magnetic beads similarly bound with a three-component association product were washed once each with ⁇ 1 SSC, ⁇ 0.1 SSC and ⁇ 0.01 SSC, followed by adding S1 Nuclease solution (Takara Bio Inc.) in the same manner as Example 1 and allowing to react, adding 30 ⁇ L of TE buffer to the resulting reaction solutions, recovering the supernatant after collecting the magnetic beads on the side of the container, irradiating with light at the excitation wavelength of TAMRA, and measuring the aforementioned supernatant by fluorescence intensity distribution analysis (FIDA).
• S1 Nuclease solution Tikara Bio Inc.
• S1 Nuclease solution (Takara Bio Inc.) was added to washed magnetic beads and allowed to react in the same manner as Example 1 and 30 ⁇ L of TE buffer were added to the resulting reaction solution, followed by recovering the supernatant after collecting the magnetic beads on the side of the container, irradiating with light at the excitation wavelength of TAMRA, and measuring the aforementioned supernatant by fluorescence correlation spectroscopy (FCS) in the same manner as Example 1 with the exception of not adding a target nucleic acid molecule to the sample solution for use as a control.
• FCS fluorescence correlation spectroscopy
• FIG. 3 The results of using FCS to measure the number of molecules of the fluorescent substance TAMRA released from the magnetic beads recovered from each sample solution are shown in FIG. 3 .
• the treatment used to release TAMRA is plotted on the horizontal axis, while the number of fluorescent molecules analyzed using FCS is plotted on the vertical axis.
• “S1 nuclease without let-7a” indicates the results of the sample solution to which the target nucleic acid molecule was not added.
• the release of a first marker from a solid phase carrier was carried out in the method for detecting a target nucleic acid molecule in still another aspect of the present invention by heating under alkaline conditions or heating under low salt concentration conditions.
• samples solutions containing a target nucleic acid molecule, first nucleic acid probe and second nucleic acid probe were prepared in the same manner as Example 1 with the exception of using, as the target nucleic acid molecule, a synthetic RNA composed of a base sequence homologous to let-7a at a final concentration of 10 nM or 0 nM, and after denaturing followed by hybridization (association), covalent bonds were formed by irradiating with ultraviolet light.
• 10 ⁇ L of magnetic beads in the form of Dynabeads (trade name: Dynabeads MyOne Streptavidin, Invitron Ltd.) were added to each sample solution followed by incubating for 15 minutes at room temperature. Subsequently, the magnetic beads were washed once each with ⁇ 1 SSC, ⁇ 0.1 SSC and ⁇ 0.01 SSC.
• “Water/0 nM” and “Water/10 nM” indicate results in the case of heating magnetic beads prepared from a sample solution having a target nucleic acid molecule concentration of 0 nM or 10 nM in water
• “1 mM NaOH/0 nM” and “1 mM NaOH/10 nM” indicate results in the case of heating magnetic beads prepared from a sample solution having a target nucleic acid molecule concentration 0 nM or 10 nM in 1 mM NaOH
• “10 mM NaOH/0 nM” and “10 mM NaOH/10 nM” indicate results in the case of heating magnetic beads prepared from sample solution having a target nucleic acid molecule concentration of 0 nM or 10 nM in 10 mM NaOH.
• TAMRA fluorescent molecules
• the release of a first marker from a solid phase carrier was carried out by heating in the method for detecting a target nucleic acid molecule in still another aspect of the present invention.
• samples solutions containing a target nucleic acid molecule, first nucleic acid probe and second nucleic acid probe were prepared in the same manner as Example 1 with the exception of using, as the target nucleic acid molecule, a synthetic RNA composed of a base sequence homologous to let-7a at a final concentration of 10 nM or 0 nM, and after denaturing followed by hybridization (association), covalent bonds were formed by irradiating with ultraviolet light.
• FIG. 5 The results of using FIDA to measure the number of molecules of the fluorescent substance TAMRA released from the magnetic beads recovered from each sample solution by heating are shown in FIG. 5 .
• the solution conditions during heating are plotted on the horizontal axis, while the number of fluorescent molecules analyzed using FIDA is plotted on the vertical axis.
• “Heat” indicates results in the case of heating at 90° C.
• “RT” indicates results in the case of not heating. In the case of not heating, hardly any fluorescent molecules were detected under all solution conditions. On the other hand, in the case of heating at 90° C., fluorescent molecules were detected under all conditions.
• a target nucleic acid molecule bound to a labeled nucleic acid probe is separated and recovered from free labeled nucleic acid probe followed by measuring in the state of being separated from a solid phase carrier. Consequently, use of the method for detecting a target nucleic acid molecule in an aspect of the present invention makes it possible to detect target nucleic acid molecules with high accuracy without being affected by free labeled probe or a solid phase carrier.
• Target nucleic acid molecule 2 : first nucleic acid probe, 2 a : fluorescent substance, 2 b: CNV K, 3 : second nucleic acid probe, 3 a : biotin, 3 b: CNV K, 4 : avidin beads

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• 2013
  • 2013-02-08 WO PCT/JP2013/053080 patent/WO2013140890A1/en active Application Filing
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